ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin

Abstract

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

Figure 1.

The p21 expression induction after ATM inhibition. CLL cells were pre-treated with ATM inhibitor (KU55933; 10 μM) for 1 h and exposed to fludarabine (3.6 μg/mL) or doxorubicin (0.25 μg/mL) for 24 h. The level of p21 expression was determined by real-time PCR in comparison with untreated control set to 100%. Sample’s C_t values were subjected to 2^−ΔΔCt analysis.

Figure 2.

The p21 expression induction in individual genetic groups. Medians of p21 expression and the significance (Kruskal-Wallis ANOVA test) related to wt group were following: (A) after doxorubicin treatment – wt: 815%; del-ATM: 731%; non-significant (NS); mut-ATM: 182%, P<0.001; def-TP53: 250%, P<0.001. (B) after fludarabine treatment – wt: 718%; del-ATM: 639%, NS; mut-ATM: 617%, NS; def-TP53: 190%, P<0.001.

Figure 3.

CLL cell viability after doxorubicin and fludarabine exposure. Significance (two-factorial ANOVA with subsequent Dunnett post-hoc test) related to wt group was: (A) after doxorubicin treatment -del-ATM: non-significant (NS), mut-ATM: P<0.001; def-TP53: P<0.001; (B) after fludarabine treatment -del-ATM: NS; mut-ATM: NS, def-TP53: P<0.001. Vertical lines represent 95% confidence intervals.

Figure 4.

Time to first treatment analysis limited to patients with unmutated IGHV. Mut-ATM and del-ATM groups related to wt patients: both P=0.04. Kaplan-Meier curves were created and statistically evaluated using the GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).